Reference standard blood serum for the calibration of automatic blood serum analyzing apparatus

ABSTRACT

A FREEZE-DRIED BLOOD SERUM IS PROVIDED FOR USE AS A REFERENCE STANDARD IN THE CALIBRATION OF AUTOMATIC BLOOD SERUM ANALYZING APPARATUS, COMPRISING A HUMAN SERUM IN WHICH THE LEVELS OF COMCENTRATION OF ITS CONSTITUENTS HAVE BEEN PRECISELY ADJUSTED, AND WHICH IS SPECIALLY PROCESSED FOR USE IN APPARATUS FOR THE SIMULTANEOUS ANALYSIS OF THE CONSTITUENTS OF EACH OF A SUCCESSION OF BLOOD SERUM SAMPLES. THE REFERENCE STANDARD BLOOD SERUM OF THE INVENTION CONTAINS TRIHYDROXYMETHYLAMINE (TRIS) CARBONATE SO AS TO PERMIT IT TO BE RECONSTITUTED JUST PRIOR TO USE WITH DISTILLED WATER SO AS TO RESTORE A PREDETERMINED CARBON DIOXIDE LEVEL IN THE REFERENCE SERUM. ALTERNATELY, THE REFERENCE STANDARD BLOOD SERUM CONTAINS. TRIS, AND IS RECONSTITUTED WITH DISTILLED WATER AND CARBON DIOXIDE.

United States Patent 3,629,142 REFERENCE STANDARD BLOOD SERUM FOR THECALIBRATION OF AUTOMATIC BLOOD SERUM ANALYZING APPARATUS Edward P.Marbach, 4607 Marwood Drive, Los Angeles, Calif. 90065 No Drawing. FiledDec. 8, 1969, Ser. No. 883,286 Int. Cl. G01n 31/22, 33/16 US. Cl.252-408 5 Claims ABSTRACT OF THE DISCLOSURE A freeze-dried blood serumis provided for use as a reference standard in the calibration ofautomatic blood serum analyzing apparatus, comprising a human serum inwhich the levels of concentration of its constituents have beenprecisely adjusted, and which is specially processed for use inapparatus for the simultaneous analysis of the constituents of each of asuccession of blood serum samples. The reference standard blood serum ofthe invention contains trihydroxymethylamine (Tris) carbonate so as topermit it to be reconstituted just prior to use with distilled water soas to restore a predetermined carbon dioxide level in the referenceserum. Alternately, the reference standard blood serum contains Tris,and is reconstituted with distilled water and carbon dioxide.

BACKGROUND OF THE INVENTION Various types of apparatus have beendeveloped in recent years for the automatic analysis of human bloodserum, and which are capable of making rapid simultaneous analyticaldeterminations on a precise quantitative basis of various components ofeach of a series of blood serum samples successively introduced into theapparatus. Biochemical determinations may be made, for example, by suchapparatus, of the following constituents of the blood serum samples:albumin, alkaline phosphatase, bilirubin, calcium, chloride,cholesterol, carbon dioxide content, creatinine, glucose, lacticdehydrogenase (LDH), inorganic phosphorus, potassium, sodium, totalprotein, transaminase (SGOT), urea nitrogen, uric acid, creatinephosphokinase (CPK).

The aforesaid apparatus may be of the type described, for example, inthe Skeggs et a1. Pat. 3,241,432 which issued Mar. 22, 1966, and whichis assigned to the Technicon Instruments Corporation of Chauncey, N.Y.The Skeggs apparatus, for example, is capable of making simultaneously amultiplicity of different analyses of a blood serum sample, with respectto the constituents referred to, for example, in the precedingparagraph, and of rapidly repeating the analyses on successive samples.

In the operation of the apparatus described in the Skeggs patent, forexample, a series of individual separate blood serum samples aresuccessively introduced into the machine, and are formed into a streamwhich subsequently is divided into a plurality of separate streams,depending upon the number of separate tests to be made. Each of thestreams is separately treated for quantitative analysis with respect tothe particular constituents, and the results of the analyses arerecorded, for example, on a chart of a recorder, or are otherwise storedin any prescribed correlated form.

As is well known, the determination of the carbon dioxide content of theblood is of great importance in many clinical procedures. The aforesaidapparatus is capable of determining the carbon dioxide content of thetest sample. Therefore, it is important for the reference standard bloodserum to include a predetermined carbon dioxide content so that it maybe used to calibrate the apparatus for that particular determination.However, it

3,629,142 Patented Dec. 21, 1971 has been found that in thefreeze-drying of the blood serum for reference standard purposes, asubstantial amount of carbon dioxide content of the serum is lost in theform of gaseous CO In order to calibrate apparatus of the type describedin the aforesaid patent, a reference standard blood serum sample is usedwhose constituents have precisely predetermined values. It is necessaryfor such reference standard serums to be capable of being stored forlong periods of time without deterioration, and for that reason it hasbeen the practice to freeze-dry them. It has also been the prior artpractice to reconstitute the reference standard serum before use bymeans, for example, of a separate aqueous solution of ammoniumbicarbonate, to restore the carbon dioxide level to a predeterminedvalue.

However, a problem which has arisen when the abovedescribed prior artreference standard serum is used is that a separate vial of a measuredamount of the ammonium bicarbonate solution had to be provided, andreliance had to be placed on the ultimate user that the reconstitutingsolution would be added in the proper quantity to the reference standardserum. This makes the reliability of the results of the apparatusdependent upon the ultimate user.

Moreover, when the prior reference standard serum is to be used fordifferent analytical procedures, different levels of carbon dioxidecontents are required, and this calls for different concentrations ofthe reconstituting solution. In such cases, different vials of differentconcentrations of the ammonium bicarbonate solution must be providedwith the prior art reference standard blood serum, and there is noassurance that the ultimate user would use the proper vial of thereconstituting solution for the appropriate procedure.

The prior art provision of the separate aqueous solution of the ammoniumbicarbonate for the reconstitution of the freeze-dried serum just priorto use is necessary, because the reconstituted prior art serum was foundto be stable only for a few days, and then only if stored inrefrigeration between, for example, 2 and 8 centigrade. It was found,for example, that storage of the reconstituted prior art reference serumat room temperature tended to alter the SGOT and LDH value in thereconstituted serum.

Also the ammonium bicarbonate could not be intermixed into the prior artreference serum originally in the laboratory because it is completelylost during freeze drying.

An important object of the present invention is to provide a referencestandard serum to which a carbonate or bicarbonate ofTris(hydroxymethyl) aminomethane is added prior to when it is in itsfreeze-dried state, and which may be reconstituted merely by addingdistilled water. The Tris carbonate has no effect on any of theconstituents of the reference standard serum and does not affect thestability of its enzyme constituents in any way, even over longintervals of time. The use of Tris carbonate takes the concentrationcontrol of the reconstituting element out of the hands of the ultimateuser, and into the laboratory where it can be established precisely andunder optimum environmental conditions. The chemical name is2-amino-2-(hydroxymethyl)-1,3-propandiol. The formula is It has beenfound that the use of the Tris carbonate reference standard serum of thepresent invention results in a reconstituted serum by the addition ofdistilled water, which functions with all the efficacy of the properlyre constituted prior art ammonium bicarbonate reference standard serum.The reference standard serum of the present invention has no tendency toplug the apparatus in which it is used, and it is clear and colorless.so that it does not affect any of the colorimeter or other tests carriedout by the apparatus. The use of Tris carbonate in the referencestandard serum of the invention results in a proper pH, so that thestability of enzymes such as lactic dehydrogenase and transaminase, isin no way affected.

An important feature of the present invention is that the addition of ameasured amount of Tris carbonate to the reference standard serumpermits the carbonate dioxide content to be restored to anypredetermined level upon reconstitution of the serum by the addition ofdistilled water, and as determined by the concentration of the Triscarbonate, depending upon the particular analytical procedure in whichthe reference standard serum is to be used.

The freeze-dried serum used in the practice of the invention can beprepared by any conventional method of freeze-drying whereby human serumis dried from a frozen state under high vacuum; wherein ice, or otherfrozen solvents, rapidly sublime to yield a porous solid. The resultingfreeze-dried reference standard serum has been found to remain stablefor over two years at temperatures ranging from 2 C. to C. In thepractice of the invention, a quantity of Tris carbonate is added to theserum prior to freeze-drying; and the freeze-dried serum is subsequentlyreconstituted by the addition of distilled water prior to use.Alternately, Tris itself is added to the serum and carbon dioxide gas isbubbled into the serum prior to freeze-drying.

This reconstituting of the serum is necessary, as explained above, inorder to increase the carbon dioxide content of the reconstituted serumto a predetermined level. as determined by the particular analyticalprocedure in which the serum is to be used for calibrating purposes. Thereconstituted serum can be used directly for the calibration and controlof the aforesaid apparatus.

The Tris carbonate is ideally suited as a component of the referencestandard blood serum in that it does not affect in any way the preciselypredetermined values of any of the constituents of the referencestandard blood serum. Also, it imparts a normal pH to the serum so as toprovide for the stability of its constituents, and particularly theenzymes, as mentioned above.

As explained above, the Tris carbonate serves to produce a carbondioxide content in the reconstituted reference standard serum which issuitable for standardizing and calibrating the test equipment. As alsopreviously stated, the Tris carbonate is colorless and has no turbidcharacteristics as would affect any of the tests made by the aforesaidapparatus.

The following specific example is intended to illustrate a particularreference serum illustrative of the practice of the invention.

As a first step in the preparation of the reference serum of theinvention, selected samples of human blood serum were pooled. Then theTris carbonate was added in sufficient amount to provide the desiredcarbon dioxide level in the reconstituted serum. For example, the Triscarbonate was formed by adding Dry Ice, as a source of carbon dioxide,to Tris. In a particular example, 65 grams of Tris was placed in 100 cc.of water, a quantity of Dry Ice was added, and the solution was allowedto stand overnight to permit the Tris carbonate to precipitate. The Triscarbonate was then dissolved in water, and added to the serum in aproportion of 7 grams of Tris carbonate per liter of serum, or in otherproportions depending upon the exact level the carbon dioxideconstituent of the serum is to be reconstituted. A volume of 50milliliters of the serum and Tris carbonate mixture was thenfreeze-dried by a known freeze-drying process.

The freeze-dried serum, prior to use, was reconstituted by adding 50milliliters of distilled water to a bottle containing the serum. Theresulting mixture was slightly agitated by rotation of the bottle sothat the serum was completely dissolved. The reconstituted pool was thenanalyzed by known procedures.

One specific sample of the reconstituted serum had the followingconstituent values which are listed herein merely by way of example:

Constituent: Value Glucose 230 mg./l00 ml. Blood urea nitrogen 69 mg./ml. Potassium 5.4 m. eq./liter Sodium 149 m. eq./liter Carbon dioxide 30m. eq./liter Chloride 113 m. eq./liter Alkaline phosphatase 25 KA unitsTransaminase (SGOT) 93 KA units Calcium 10.2 mg./l00 mil Phosphorous 4.8mg./1()0 mil Uric acid 4.2 mg./l00 mil Cholesterol 200 mg./l00 mil Totalprotein 6.6 gm./l00 mil Albumin 3.9 gm./l00 mil Bilirubin 2.0 mg./l00mil Lactic dehydrogenase 220 Wacker units What is claimed is:

1. A freeze-dried blood serum reference standard comprising blood serumhaving constituents of known values and a carbonate or bicarbonateTristhydroxymethyl) aminomethane.

2. The blood serum of claim 1, in which the concentration of theaforesaid aminomethane in the serum is such that when the serum isreconstituted, the carbon dioxide content of the reconstituted serum isin a range extending from approximately 12 to approximately 35 m.eq./liter.

3. The blood serum of claim 1, reconstituted by the addition ofdistilled water.

4. The method of providing an accurate blood serum reference standardhaving a standard carbon dioxide content for use in multi-automatedanalytical procedures which comprises reconstituting freeze-dried bloodserum containing a quantity of a carbonate or bicarbonate ofTris(hydroxymethyl)aminomethane with distilled water so that the carbondioxide content of the reconstituted serum may be increased to apredetermined level.

5. The method set forth in claim 4, in which the concentration of theaforesaid aminomethane is such that the carbon dioxide content of thereconstituted serum is in a range extending from approximately 12 toapproximately 35 m. eq./liter.

References Cited UNITED STATES PATENTS 3,466,249 9/1969 Anderson 252-408JOHN T. GOOLKASIAN, Primary Examiner M. E. MCCAMISH, Assistant ExaminerUS. Cl. X.R. 23-230 B

